Comparative Oncogenomics Identifies Novel Regulators and Clinical Relevance of Neural Crest Identities in Melanoma

Cancers often resurrect embryonic molecular programs to promote disease progression. In melanomas, which are tumors of the neural crest (NC) lineage, a molecular signature of the embryonic NC is often reactivated. These NC factors have been implicated in promoting pro-tumorigenic features like proliferation, migration and therapy resistance. However, the molecular mechanisms that establish and maintain NC identities in melanomas are largely unknown. Additionally, whether the presence of a NC identity has any clinical relevance for patient melanomas is also unclear. Here, using comparative genomic approaches, I have a) identified a novel role for GDF6-activated BMP signaling in reawakening a NC identity in melanomas, and b) identified a NC signature as a clinical predictor of melanoma progression. Like the genomes of many solid cancers, melanoma genomes have widespread copy number variations (CNV) harboring thousands of genes. To identify disease-promoting drivers amongst such huge numbers of genes, I used a comparative oncogenomics approach with zebrafish and human melanomas. This approach led to the identification of a recurrently amplified oncogene, GDF6, that acts via BMP signaling to invoke NC identities in melanomas. In maintaining this identity, GDF6 represses the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, allowing melanoma cells to remain undifferentiated and survive. Functional analysis in zebrafish embryos indicated a role of GDF6 in blocking melanocyte differentiation, suggesting that the developmental function of GDF6 is reiterated in melanomas. In clinical assessments, a major fraction of patient melanomas expressed high GDF6, and its expression correlated with poor patient survival. These studies provide novel insights into regulation of NC identities in melanomas and offer GDF6 and components of BMP pathway as targets for therapeutic intervention. In additional studies, I wanted to test whether a broader NC identity in melanomas had any clinical relevance. In these studies, I performed transcriptome analysis of zebrafish melanomas and derived a 15-gene NC signature. This NC gene signature positively correlated with the expression of SOX10, a known NC marker in human melanomas. Patients whose melanomas expressed this signature showed poor overall survival. These findings identify an important predictive signature in human melanomas and also illuminate the clinical importance of NC identity in this disease

CNF Satisfiability in a Subspace and Related Problems

We introduce the problem of finding a satisfying assignment to a CNF formula that must further belong to a prescribed input subspace. Equivalent formulations of the problem include finding a point outside a union of subspaces (the Union-of-Subspace Avoidance (USA) problem), and finding a common zero of a system of polynomials over ?? each of which is a product of affine forms. We focus on the case of k-CNF formulas (the k-Sub-Sat problem). Clearly, k-Sub-Sat is no easier than k-SAT, and might be harder. Indeed, via simple reductions we show that 2-Sub-Sat is NP-hard, and W[1]-hard when parameterized by the co-dimension of the subspace. We also prove that the optimization version Max-2-Sub-Sat is NP-hard to approximate better than the trivial 3/4 ratio even on satisfiable instances. On the algorithmic front, we investigate fast exponential algorithms which give non-trivial savings over brute-force algorithms. We give a simple branching algorithm with running time (1.5)^r for 2-Sub-Sat, where r is the subspace dimension, as well as an O^*(1.4312)? time algorithm where n is the number of variables. Turning to k-Sub-Sat for k ? 3, while known algorithms for solving a system of degree k polynomial equations already imply a solution with running time ? 2^{r(1-1/2k)}, we explore a more combinatorial approach. Based on an analysis of critical variables (a key notion underlying the randomized k-SAT algorithm of Paturi, Pudlak, and Zane), we give an algorithm with running time ? {n choose {?t}} 2^{n-n/k} where n is the number of variables and t is the co-dimension of the subspace. This improves upon the running time of the polynomial equations approach for small co-dimension. Our combinatorial approach also achieves polynomial space in contrast to the algebraic approach that uses exponential space. We also give a PPZ-style algorithm for k-Sub-Sat with running time ? 2^{n-n/2k}. This algorithm is in fact oblivious to the structure of the subspace, and extends when the subspace-membership constraint is replaced by any constraint for which partial satisfying assignments can be efficiently completed to a full satisfying assignment. Finally, for systems of O(n) polynomial equations in n variables over ??, we give a fast exponential algorithm when each polynomial has bounded degree irreducible factors (but can otherwise have large degree) using a degree reduction trick

Disseminated tuberculosis in elderly-latent activation mimics metastasis: a case study

Tuberculosis (TB) is an infectious disease caused by mycobacterium species, that affects the lungs and involvement of the musculoskeletal system is not uncommon. Two or more systemic non-contiguous spread is termed as disseminated TB, and its additional involvement of lungs constitute the diagnosis of miliary TB (MTB). Immuno-suppressive conditions including malnutrition and immune-senescence in elderly can predispose to reactivation of latent TB and wide spread dissemination of the bacilli. Screening for dormant TB infection remains an impractical task and its diagnostic confirmation remains a challenge. With raising elderly population, there is a need for a high index of suspicion in the diagnosis of disseminated TB and its management, as delayed approach has increased mortality. Atypical presentation of disseminated TB with concomitant vertebral collapse and extensive lymphadenopathy needs exclusion of metastatic disease although the old nemesis of TB remains a differential diagnosis. We present one such case in a 71 years old lady to highlight the atypical presentation of disseminated TB. The disease presentation had spino-dermal pattern with lympho-reticular involvement including asymptomatic lesions bilaterally in the adrenal glands lesions

Regulation of zebrafish melanocyte development by ligand-dependent BMP signaling

Preventing terminal differentiation is important in the development and progression of many cancers including melanoma. Recent identification of the BMP ligand GDF6 as a novel melanoma oncogene showed GDF6-activated BMP signaling suppresses differentiation of melanoma cells. Previous studies have identified roles for GDF6 orthologs during early embryonic and neural crest development, but have not identified direct regulation of melanocyte development by GDF6. Here, we investigate the BMP ligand gdf6a, a zebrafish ortholog of human GDF6, during the development of melanocytes from the neural crest. We establish that the loss of gdf6a or inhibition of BMP signaling during neural crest development disrupts normal pigment cell development, leading to an increase in the number of melanocytes and a corresponding decrease in iridophores, another neural crest-derived pigment cell type in zebrafish. This shift occurs as pigment cells arise from the neural crest and depends on mitfa, an ortholog of MITF, a key regulator of melanocyte development that is also targeted by oncogenic BMP signaling. Together, these results indicate that the oncogenic role ligand-dependent BMP signaling plays in suppressing differentiation in melanoma is a reiteration of its physiological roles during melanocyte development

Developing anti-GDF6 therapeutics for treatment of advanced melanoma

Melanoma, the leading cause of skin cancer death in the U.S., is increasing in incidence. Targeted therapies have been approved for treatment of advanced melanoma, but few patients experience extended survival benefit. In order to combat poor outcomes, new therapeutic targets are needed. Using cross-species oncogenomic analyses, our lab has identified a novel melanoma driver, Growth differentiation factor 6 (GDF6), a secreted bone morphogenetic protein (BMP) ligand that is amplified and overexpressed in human melanomas. Functional analyses show GDF6 acts via the BMP-SMAD1 pathway as a pro-survival factor in melanomas. Inhibiting GDF6 or the BMP pathway using shRNAs or the small molecule inhibitor, DMH1, induces melanoma cell death thereby abrogating melanoma growth in mouse xenografts. These results suggest GDF6 is an optimal target melanoma therapy. In order to better understand the dynamics of GDF6 signaling in melanoma cells, we are currently investigating the effect of exogenous GDF6 on cells with inhibited GDF6 expression to determine the required concentration to activate SMAD1 signaling and rescue viability. As GDF6 is a secreted ligand, we proposed developing antibodies to block the GDF6 interaction at its receptor, thereby inhibiting signaling. In collaboration with MassBiologics, we have generated a panel of monoclonal antibodies targeting GDF6. To identify antibodies capable of blocking GDF6 activity, we have devised a series of assays to eliminate antibodies from the panel. First, candidates are screened for affinity to GDF6. Second, candidates are screened for ability to block interaction between GDF6 and its receptor. Third, candidates are evaluated for ability to inhibit downstream signaling via SMAD1 pathway. After selection of final candidates, we will use a xenograft model to determine ability to inhibit melanoma growth in vivo. Currently, we have identified antibodies that are able to recognize GDF6 via western blot, and are proceeding to screen these antibodies for anti-GDF6 activity

Identification of GDF-6 blocking antibodies as anti-melanoma therapeutics

Through comparative oncogenomic studies and functional analyses, we have identified the bone morphogenetic protein (BMP) factor GDF6 as a new melanoma oncogene. The secreted, carboxy-terminal portion of GDF6 is the active form that binds to cell-surface receptors to initiate BMP signaling. Targeted antibodies directed against secreted proteins are a proven therapeutic modality in several diseases. To develop therapeutic antibodies against the active form of GDF6, we generated a panel of monoclonal antibodies. Due to the high similarity of human and mouse GDF6 proteins, the C-terminal GDF6 protein was expressed as bacterial recombinant protein with fusion tags to enhance immunogenicity. The Expresso Screening System (Lucigen) was used to select fusion tags, and MBP and SlyD were chosen for optimal protein solubility and purification recovery. Ten CD1 mice were immunized with GDF6-MBP fusion protein and robust immune responses were observed in all animals after 5 immunizations. Animals were sacrificed for hybridoma fusion, and hybridoma clones were screened by ELISA using GDF6-SlyD fusion protein to select clones with specific binding activity to GDF6. Over 70 monoclonal antibodies were identified with strong reactivity to GDF6, and a subset has been shown to recognize the endogenous, secreted form of GDF6 via western blot. These antibodies will be screened for their activity to block GDF6 binding to melanoma cells and ability to inhibit downstream signaling using both in vitro assays and in vivo xenograft models

Ligand-activated BMP signaling inhibits cell differentiation and death to promote melanoma

Oncogenomic studies indicate that copy number variation (CNV) alters genes involved in tumor progression; however, identification of specific driver genes affected by CNV has been difficult, as these rearrangements are often contained in large chromosomal intervals among several bystander genes. Here, we addressed this problem and identified a CNV-targeted oncogene by performing comparative oncogenomics of human and zebrafish melanomas. We determined that the gene encoding growth differentiation factor 6 (GDF6), which is the ligand for the BMP family, is recurrently amplified and transcriptionally upregulated in melanoma. GDF6-induced BMP signaling maintained a trunk neural crest gene signature in melanomas. Additionally, GDF6 repressed the melanocyte differentiation gene MITF and the proapoptotic factor SOX9, thereby preventing differentiation, inhibiting cell death, and promoting tumor growth. GDF6 was specifically expressed in melanomas but not melanocytes. Moreover, GDF6 expression levels in melanomas were inversely correlated with patient survival. Our study has identified a fundamental role for GDF6 and BMP signaling in governing an embryonic cell gene signature to promote melanoma progression, thus providing potential opportunities for targeted therapy to treat GDF6-positive cancers

A sustained ocean observing system in the Indian Ocean for climate related scientific knowledge and societal needs

© The Author(s), 2019. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Hermes, J. C., Masumoto, Y., Beal, L. M., Roxy, M. K., Vialard, J., Andres, M., Annamalai, H., Behera, S., D'Adamo, N., Doi, T., Peng, M., Han, W., Hardman-Mountford, N., Hendon, H., Hood, R., Kido, S., Lee, C., Lees, T., Lengaigne, M., Li, J., Lumpkin, R., Navaneeth, K. N., Milligan, B., McPhaden, M. J., Ravichandran, M., Shinoda, T., Singh, A., Sloyan, B., Strutton, P. G., Subramanian, A. C., Thurston, S., Tozuka, T., Ummenhofer, C. C., Unnikrishnan, A. S., Venkatesan, R., Wang, D., Wiggert, J., Yu, L., & Yu, W. (2019). A sustained ocean observing system in the Indian Ocean for climate related scientific knowledge and societal needs. Frontiers in Marine Science, 6, (2019): 355, doi: 10.3389/fmars.2019.00355.The Indian Ocean is warming faster than any of the global oceans and its climate is uniquely driven by the presence of a landmass at low latitudes, which causes monsoonal winds and reversing currents. The food, water, and energy security in the Indian Ocean rim countries and islands are intrinsically tied to its climate, with marine environmental goods and services, as well as trade within the basin, underpinning their economies. Hence, there are a range of societal needs for Indian Ocean observation arising from the influence of regional phenomena and climate change on, for instance, marine ecosystems, monsoon rains, and sea-level. The Indian Ocean Observing System (IndOOS), is a sustained observing system that monitors basin-scale ocean-atmosphere conditions, while providing flexibility in terms of emerging technologies and scientificand societal needs, and a framework for more regional and coastal monitoring. This paper reviews the societal and scientific motivations, current status, and future directions of IndOOS, while also discussing the need for enhanced coastal, shelf, and regional observations. The challenges of sustainability and implementation are also addressed, including capacity building, best practices, and integration of resources. The utility of IndOOS ultimately depends on the identification of, and engagement with, end-users and decision-makers and on the practical accessibility and transparency of data for a range of products and for decision-making processes. Therefore we highlight current progress, issues and challenges related to end user engagement with IndOOS, as well as the needs of the data assimilation and modeling communities. Knowledge of the status of the Indian Ocean climate and ecosystems and predictability of its future, depends on a wide range of socio-economic and environmental data, a significant part of which is provided by IndOOS.This work was supported by the PMEL contribution no. 4934